Hypertension, or high blood pressure, is a common disease affecting 50 million Americans and contributing to over 200,000 deaths annually from stroke, myocardial infarction, and end-stage renal disease. The disease is multifactorial and numerous genetic and nongenetic components, such as salt intake, age, diet, and body mass, are suspected to contribute. A specific cause of hypertension can typically be identified in only a small percentage of patients. Other patients with abnormally high blood pressure of unknown cause are said to have essential hypertension.
The existence of a genetic component to hypertension is known from twin studies, which have revealed a greater concordance of blood pressure in monozygotic twins that in dizygotic twins. Similarly, biological siblings have show greater concordance of blood pressure than adoptive siblings raised in the same household. Such studies have suggested that up to about 40% of the variations in blood pressure in the population are genetically determined.
There is a substantial pool of candidate genes that may contribute to the genetic component of hypertension. Because blood pressure is determined by the product of cardiac output and vascular resistance, candidate genes may act through either pathway. Physiologic pathways which are know to influence these parameters include the renin-angiotensin-aldosterone system, which contributes to determination of both cardiac output and vascular resistance. In this pathway, angiotensinogen, a hormone produced in the liver, is cleaved by an enzyme called renin to angiotensin I, which then undergoes further cleavage by angiotensin I-converting enzyme (ACE) to produce the active hormone angiotensin II (AII). All acts through specific AT1 receptors present on vascular and adrenal cells. Receptors present on vascular cells cause vasoconstriction of blood vessels. Receptors present on adrenal cells cause release of the hormone aldosterone by the adrenal gland. This hormone acts on the mineralocorticoid receptor to cause increase sodium reabsorption largely through a renal epithelial sodium location. Other candidate genes are those of peripheral and central adrenergic pathways, which have dominant effects on cardiac iontropy, heart rate and vascular resistance; a variety of renal ion channels and transporters, which determine net sodium absorption and hence intravascular volume; calcium channels and exchangers and nitric oxide pathways, whose activity influences vascular tone. Another candidate gene encodes atrial natriuretic factor precursor, which is cleaved to atrial natriuretic peptides, found in the heart atrium, an endocrine organ controlling blood pressure and organ volume.
For some of the above candidate genes, variant forms have been identified that occur with increased frequency in individuals with hypertension. For example, a number of the polymorphisms have been reported in the angiotensinogen gene (AGT). In one of these, an M/T substitution at position 235, the T allele occurs more frequently in individuals with hypertension suggesting that this polymorphic form is a cause of hypertension or in equilibrium dislinkage with another polymorphism that is a cause. Jeunmaitre et al., Am. J. Hum. Genet. 60, 1448-1460 (1997). Two other genes within the renin-angiotensin-aldosterone system also have variant forms correlated with specific forms of hypertension, that is, aldosterone synthase gene and the gene encoding the xcex2-subunit of the epithelial sodium channel induced by the mineralocorticoid receptor. Lifton et al., Proc. Natl. Acad. Sci. USA 92, 8548-8551(1995).
Despite these developments, only a minute proportion of the total repository of polymorphisms in candidate genes for hypertension has been identified, and the primary genetic determinants of hypertension remain unknown in most affected subjects, as does the nature of the interaction between different genetic determinants. The paucity of polymorphisms hitherto identified is due to the large amount of work required for their detection by conventional methods. For example, a conventional approach to identifying polymorphisms might be to sequence the same stretch of oligonucleotides in a population of individuals by dideoxy sequencing. In this type of approach, the amount of work increases in proportion to both the length of sequence and the number of individuals in a population and becomes impractical for large stretches of DNA or large numbers of persons.
The invention provides nucleic acids of between 10 and 100 bases comprising at least 10 contiguous nucleotides including a polymorphic site from a sequence shown in Table 1, column 8 or the complement thereof. The nucleic acids can be DNA or RNA. Some nucleic acids are between 10 and 50 bases and some are between 20 and 50 bases. The base occupying the polymorphic site in such nucleic acids can be either a reference base shown in Table 1, column 3 or an alternative base shown in Table 1, column 5. In the some nucleic acids, the polymorphic site is occupied by a base that correlates with hypertension or susceptibility thereto. Some nucleic acids contain a polymorphic site having two polymorphic forms giving rise two different amino acids specified by the two codons in which the polymorphic site occurs in the two polymorphic forms.
The invention further provides allele-specific oligonucleotides that hybridize to a nucleic acid segment shown in Table 1, column 8 or its complement, including the polymorphic site. Such oligonucleotides are useful as probes or primers.
The invention further provides methods of analyzing a nucleic acid sequence. Such methods entail obtaining the nucleic acid from an individual; and determining a base occupying any one of the polymorphic sites shown in Table 1 or other polymorphic sites in equilibrium dislinkage therewith. Some methods determine a set of bases occupying a set of the polymorphic sites shown in Table 1. In some methods, the nucleic acid is obtained from a plurality of individuals, and a base occupying one of the polymorphic positions is determined in each of the individuals. Each individual is then tested for the presence of a disease phenotype, and correlating the presence of the disease phenotype with the base, particularly hypertension.
In another aspect, the invention provides nucleic acids comprising an isolated nucleic acid sequence of Table 1, column 8 or the complement thereof, wherein the polymorphic site within the sequence or its complement is occupied by a base other than the reference base show in Table 1, column 3. Such nucleic acids are useful, for example, in expression of variant proteins or production of transgenic animals.
The invention further provides methods of diagnosing a phenotype. Such methods entail determining which polymorphic form(s) are present in a sample from a subject at one or more polymorphic sites shown in Table 1, and diagnosing the presence of a phenotype correlated with the form(s) in the subject.
The invention also provides methods of screening polymorphic sites linked to polymorphic sites shown in Table 1 for suitability for diagnosing a phenotype. Such methods entail identifying a polymorphic site linked to a polymorphic site shown in Table 1, wherein a polymorphic form of the polymorphic site shown in Table 1 has been correlated with a phenotype. One then determines haplotypes in a population of individuals to indicate whether the linked polymorphic site has a polymorphic form in equlibrium dislinkage with the polymorphic form correlated with the phenotype.